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rabbit polyclonal anti chmp5 (Thermo Fisher)

Name: rabbit polyclonal anti chmp5
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Rating: 86 (1 reviews)

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Thermo Fisher rabbit polyclonal anti chmp5
Rabbit Polyclonal Anti Chmp5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a Micro-CT and Haemotoxylin and Eosin (H&E) staining showing periskeletal overgrowth near the joint in Chmp5 Ctsk mice in comparison with Chmp5 fl/fl mice at 7 weeks of age. Dot-line representing the approximate border between the periskeletal overgrowth and bone at the femoral condyle of the knee. Images are representative of 10 animals per group. b Micro-CT images displaying bone overgrowth at the ankle and foot in Chmp5 Ctsk mice at one year of age. Images are representative of 4 animals per group. c Micro-CT, TRAP staining, and measurement of orbital distance and head width demonstrating craniofacial suture abnormalities in Chmp5 Ctsk mice at 6 weeks of age. Images are representative of 10 Chmp5 fl/fl and 11 Chmp5 Ctsk mice. d , e Confocal images mapping Ctsk + (GFP + ) progenitors and their descendants in periskeletal tissues ( d ) and the sagittal suture ( e ) in Chmp5 Ctsk/+ ;Rosa26 mTmG/+ and Chmp5 Ctsk ;Rosa26 mTmG/+ mice at 2 weeks of age. In ( d ), asterisk indicating periskeletal overgrowths; dot-line representing the approximate border between the periskeletal overgrowth and bone. Images are representative of 4 animals each group. f Flow cytometry showing strategy of sorting CD45 - ;CD31 - ;GFP + and CD45 - ;CD31 - ;GFP - skeletal progenitors from periskeletal tissues of Chmp5 Ctsk/+ ;Rosa26 mTmG/+ and Chmp5 Ctsk ;Rosa26 mTmG/+ mice at 2 weeks of age. g Quantitative PCR determining the deletion of Chmp5 in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 6 mice per group. h Quantitative PCR determining the expression of PGE2 pathway related genes Hpgd , Ptgs1 , Ptgs2 , and Abcc4 in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 6 animals per group. All data are mean ± s.d.; two-tailed Student’s t -test. Scale bars, 200 µm except 1 mm for micro-CT images.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Micro-CT and Haemotoxylin and Eosin (H&E) staining showing periskeletal overgrowth near the joint in Chmp5 Ctsk mice in comparison with Chmp5 fl/fl mice at 7 weeks of age. Dot-line representing the approximate border between the periskeletal overgrowth and bone at the femoral condyle of the knee. Images are representative of 10 animals per group. b Micro-CT images displaying bone overgrowth at the ankle and foot in Chmp5 Ctsk mice at one year of age. Images are representative of 4 animals per group. c Micro-CT, TRAP staining, and measurement of orbital distance and head width demonstrating craniofacial suture abnormalities in Chmp5 Ctsk mice at 6 weeks of age. Images are representative of 10 Chmp5 fl/fl and 11 Chmp5 Ctsk mice. d , e Confocal images mapping Ctsk + (GFP + ) progenitors and their descendants in periskeletal tissues ( d ) and the sagittal suture ( e ) in Chmp5 Ctsk/+ ;Rosa26 mTmG/+ and Chmp5 Ctsk ;Rosa26 mTmG/+ mice at 2 weeks of age. In ( d ), asterisk indicating periskeletal overgrowths; dot-line representing the approximate border between the periskeletal overgrowth and bone. Images are representative of 4 animals each group. f Flow cytometry showing strategy of sorting CD45 - ;CD31 - ;GFP + and CD45 - ;CD31 - ;GFP - skeletal progenitors from periskeletal tissues of Chmp5 Ctsk/+ ;Rosa26 mTmG/+ and Chmp5 Ctsk ;Rosa26 mTmG/+ mice at 2 weeks of age. g Quantitative PCR determining the deletion of Chmp5 in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 6 mice per group. h Quantitative PCR determining the expression of PGE2 pathway related genes Hpgd , Ptgs1 , Ptgs2 , and Abcc4 in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 6 animals per group. All data are mean ± s.d.; two-tailed Student’s t -test. Scale bars, 200 µm except 1 mm for micro-CT images.

Article Snippet: Rabbit anti-CHMP5 polyclonal antibody Mouse anti-EEA1 antibody (clone 14/EEA1, BD Bioscience) Rabbit anti-RAB7 antibody (clone D95F2, Cell Signaling Technology) Rabbit anti-RAB11 antibody (clone D4F5, Cell Signaling Technology) Rat anti-CD107a (LAMP1) antibody (clone 1D4B, BD Bioscience) Rat anti-TGN38 antibody (clone 831629, R&D Systems) Mouse anti-OXPHOS antibody cocktail (cat#ab110413, Abcam) Rabbit anti-Ki67 antibody (cat# ab15580, Abcam) Mouse anti-GAPDH antibody (clone GT239, GeneTex) Mouse anti-beta-actin antibody (clone 2F1-1, Biolegend) APC anti-mouse CD45 (clone 30-F11, Biolegend) APC anti-mouse CD31 (clone 390, Biolegend) Goat anti-Rat secondary antibody, Alexa Fluor 633 (cat# A-21094, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 633 (cat# A-21050, ThermoFisher) Goat anti-Rat secondary antibody, Alexa Fluor 488 (cat# A-11006, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 488 (cat# A-11001, ThermoFisher) Goat anti-Rat secondary antibody, Alexa Fluor 594 (cat# A-11007, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 594 (cat# A-11005, ThermoFisher) Goat anti-Rabbit secondary antibody, Alexa Fluor® 594 (cat# A-11012, ThermoFisher) In Situ Apoptosis Detection Kit (cat#ab206386, Abcam) Lysotracker™ Red DND-99 (cat# L7528, ThermoFisher) pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate (cat# P35374, ThermoFisher) Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE, cat# T669, ThermoFisher) MitoTracker™ Green FM (cat# M7514, ThermoFisher) CellROX™ Deep Red Reagent (cat# C10422, ThermoFisher) N-Acetyl-L-cysteine (cat# A7250, Sigma-Aldrich) Quercetin (cat# Q4951, Sigma-Aldrich) Dasatinib (cat# CDS023389, Sigma-Aldrich) Polyethylene glycol 400 (PEG400, Fisher Scientific) Annexin V Apoptosis Detection Kit APC (cat# 88-8007-72, ThermoFisher) APC BrdU Flow Kit (cat# 552598, BD Bioscience) InSolution™ Q-VD-OPh, Non-O-methylated (cat# 551476, Millipore) VECTASTAIN® Elite® ABC HRP Kit (Peroxidase, Rabbit IgG; cat# PK-6101, VECTOR Laboratories) Recombinant Mouse TRANCE/RANK L/TNFSF11 Protein (cat# 462-TR, R&D Systems) Recombinant Mouse M-CSF Protein (cat# 461-ML, R&D Systems) alamarBlue™ Cell Viability Reagent (cat# DAL1025, ThermoFisher) Seahorse XF Cell Mito Stress Test Kit (cat# 103015-100, Agilent) ALIZARIN red 2% solution pH 4.2 (cat# 50-317-34, Fisher scientific) Zolendronic acid monohydrate (cat# SML0223, Sigma-Aldrich) OPG-Fc (Amgen Inc.) MC3T3-E1 cells (mouse C57BL/6 calvarial fibroblasts; cat# 99072810, Sigma-Aldrich) ATDC5 cells (mouse 129 teratocarcinoma-derived osteochondral progenitors; cat# 99072806, Sigma-Aldrich)

Techniques: Micro-CT, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

a Micro-CT images showing progressive craniofacial suture and zygomatic bone (arrows) lesions with age. Images are representative of 6 mice per group at 7 weeks of age, 5 mice per group at 18 mons of age. b Representative micro-CT images displaying abnormal bone formation around the mandibular condyle of temporomandibular joint (arrow) with limited mouth-opening. n = 37 Chmp5 fl/fl , 33 Chmp5 Ctsk mice. c TRAP staining showing no osteoclasts in the periskeletal overgrowth (asterisk) in Chmp5 Ctsk mice at 2 weeks of age. TRAP + osteoclasts in the bone marrow as the positive control; dot-line representing the approximate border between the periskeletal overgrowth and bone at the femoral condyle of the knee. Images are representative of 3 animals per group. d-h Gross and micro-CT images demonstrating skeletal phenotypes after treatment with PBS, Zoledronic acid, or OPG-Fc at 2 weeks ( d-f ) or 4 weeks ( g, h ) of ages. Both arrows and arrowheads indicating periskeletal overgrowths. Images are representative of 4 ( d ), 5 ( e , f ), or 3 ( g , h ) mice per group. i Confocal images of Rosa26 mTmG/+ (Cre - ) control mice. Related to . j Unstained cells from Rosa26 mTmG/+ mice as a negative control for sorting Ctsk + skeletal progenitors. Related to . Data are mean ± s.d.; two-tailed Student’s t -test. Scale bars, 1 mm for micro-CT images, 200 µm for histological images.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Micro-CT images showing progressive craniofacial suture and zygomatic bone (arrows) lesions with age. Images are representative of 6 mice per group at 7 weeks of age, 5 mice per group at 18 mons of age. b Representative micro-CT images displaying abnormal bone formation around the mandibular condyle of temporomandibular joint (arrow) with limited mouth-opening. n = 37 Chmp5 fl/fl , 33 Chmp5 Ctsk mice. c TRAP staining showing no osteoclasts in the periskeletal overgrowth (asterisk) in Chmp5 Ctsk mice at 2 weeks of age. TRAP + osteoclasts in the bone marrow as the positive control; dot-line representing the approximate border between the periskeletal overgrowth and bone at the femoral condyle of the knee. Images are representative of 3 animals per group. d-h Gross and micro-CT images demonstrating skeletal phenotypes after treatment with PBS, Zoledronic acid, or OPG-Fc at 2 weeks ( d-f ) or 4 weeks ( g, h ) of ages. Both arrows and arrowheads indicating periskeletal overgrowths. Images are representative of 4 ( d ), 5 ( e , f ), or 3 ( g , h ) mice per group. i Confocal images of Rosa26 mTmG/+ (Cre - ) control mice. Related to . j Unstained cells from Rosa26 mTmG/+ mice as a negative control for sorting Ctsk + skeletal progenitors. Related to . Data are mean ± s.d.; two-tailed Student’s t -test. Scale bars, 1 mm for micro-CT images, 200 µm for histological images.

Techniques: Micro-CT, Staining, Positive Control, Negative Control, Two Tailed Test

a Representative fluorescence image showing fate-mapping of Dmp1 + (GFP + ) osteoprogenitors and their descendants in the tibia of Dmp1-Cre;Rosa26 mTmG/+ mice at 4 weeks of age. b , c Micro-CT analyses of trabecular and cortical bone parameters in Chmp5 Dmp1 mice compared to Chmp5 fl/fl mice. n = 5 animals per group, representative data from male mice shown, similar trend of changes found in both genders. d TRAP staining and activity assay determining osteoclastogenesis in bone marrow stromal cells of Chmp5 Dmp1 and Chmp5 fl/fl mice. n = 4 animals per group, repeated twice. Data shown as mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons. Scale bars, 200 µm.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Representative fluorescence image showing fate-mapping of Dmp1 + (GFP + ) osteoprogenitors and their descendants in the tibia of Dmp1-Cre;Rosa26 mTmG/+ mice at 4 weeks of age. b , c Micro-CT analyses of trabecular and cortical bone parameters in Chmp5 Dmp1 mice compared to Chmp5 fl/fl mice. n = 5 animals per group, representative data from male mice shown, similar trend of changes found in both genders. d TRAP staining and activity assay determining osteoclastogenesis in bone marrow stromal cells of Chmp5 Dmp1 and Chmp5 fl/fl mice. n = 4 animals per group, repeated twice. Data shown as mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons. Scale bars, 200 µm.

Techniques: Fluorescence, Micro-CT, Staining, Activity Assay, Two Tailed Test

a X-ray images demonstrating progressive cortical bone expansion (arrows) and decreased skeletal muscle volume (asterisks) in Chmp5 Dmp1 mice in comparison with Chmp5 fl/fl mice. Images are representative of 3, 3, 10, 8 animals per group for 3 weeks, 5 weeks, 10 weeks, and 1 year of ages respectively. Scale bars, 10 mm. b Gross image and measurement of femur thickness showing bone expansion in Chmp5 Dmp1 versus Chmp5 fl/fl mice at 10 weeks of age. n = 13 Chmp5 fl/fl and 12 Chmp5 Dmp1 mice. Scale bar, 1 mm. c Micro-CT analysis displaying cortical bone expansion and decreased trabecular bone mass in Chmp5 Dmp1 relative to Chmp5 fl/fl mice at 10 weeks of age. n = 6 Chmp5 fl/fl and 5 Chmp5 Dmp1 male mice, similar trend of changes found in both genders. Scale bars, 1 mm. d H&E staining showing bone expansion at the endosteum (arrows) and decreased trabecular bone mass (right) in Chmp5 Dmp1 mice. The left panels showing midshaft and the right panels showing metaphysis of the femur bone. Images are representative of 3 mice per group. Scale bars, 0.5 mm. e Micro-CT images demonstrating craniofacial bone expansion (arrows) in Chmp5 Dmp1 relative to Chmp5 fl/fl mice at 10 weeks of age. n = 6 Chmp5 fl/fl and 5 Chmp5 Dmp1 animals. f Hindlimb abduction of Chmp5 Dmp1 mice in comparison with Chmp5 fl/fl littermate controls at 10 weeks of age. More than 20 animals examined for each group. g Skeletal muscle mass (quadriceps and gastrocnemius, n = 4 per group, male mice), four-limb handing time ( n = 10 per group, pooled data from male and female mice), and forelimb pull strength ( n = 6 each group, male mice) in Chmp5 Dmp1 mice compared to Chmp5 fl/fl mice at 10-12 weeks of age. For skeletal muscle mass and forelimb pull strength, similar trend of changes was found in both genders. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a X-ray images demonstrating progressive cortical bone expansion (arrows) and decreased skeletal muscle volume (asterisks) in Chmp5 Dmp1 mice in comparison with Chmp5 fl/fl mice. Images are representative of 3, 3, 10, 8 animals per group for 3 weeks, 5 weeks, 10 weeks, and 1 year of ages respectively. Scale bars, 10 mm. b Gross image and measurement of femur thickness showing bone expansion in Chmp5 Dmp1 versus Chmp5 fl/fl mice at 10 weeks of age. n = 13 Chmp5 fl/fl and 12 Chmp5 Dmp1 mice. Scale bar, 1 mm. c Micro-CT analysis displaying cortical bone expansion and decreased trabecular bone mass in Chmp5 Dmp1 relative to Chmp5 fl/fl mice at 10 weeks of age. n = 6 Chmp5 fl/fl and 5 Chmp5 Dmp1 male mice, similar trend of changes found in both genders. Scale bars, 1 mm. d H&E staining showing bone expansion at the endosteum (arrows) and decreased trabecular bone mass (right) in Chmp5 Dmp1 mice. The left panels showing midshaft and the right panels showing metaphysis of the femur bone. Images are representative of 3 mice per group. Scale bars, 0.5 mm. e Micro-CT images demonstrating craniofacial bone expansion (arrows) in Chmp5 Dmp1 relative to Chmp5 fl/fl mice at 10 weeks of age. n = 6 Chmp5 fl/fl and 5 Chmp5 Dmp1 animals. f Hindlimb abduction of Chmp5 Dmp1 mice in comparison with Chmp5 fl/fl littermate controls at 10 weeks of age. More than 20 animals examined for each group. g Skeletal muscle mass (quadriceps and gastrocnemius, n = 4 per group, male mice), four-limb handing time ( n = 10 per group, pooled data from male and female mice), and forelimb pull strength ( n = 6 each group, male mice) in Chmp5 Dmp1 mice compared to Chmp5 fl/fl mice at 10-12 weeks of age. For skeletal muscle mass and forelimb pull strength, similar trend of changes was found in both genders. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Techniques: Micro-CT, Staining, Two Tailed Test

a Cell number counting and AlamarBlue assay determining cell proliferation in Chmp5 Ctsk and Chmp5 Ctsk/+ skeletal progenitors at 2 weeks of age. n = 3 replicates per group per time-point; experiments repeated 3 times using cells from 3 mice. b Gene set enrichment analysis (GSEA) of transcriptome data showing positive enrichment of genes associated with cell senescence in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Enriched gene list referring to Supplementary Table 1 . n = 3 mice per group for RNA-sequencing analysis. c Gross image demonstrating phenotypes of accelerating senescence in Chmp5 Ctsk mice compared with Chmp5 Ctsk/+ mice at one year of age. Images are representative of 5 animals per group. d Cell number counting determining cell proliferation in ATDC5 cells with or without Chmp5 deletion by lentiviral CRISPR/CAS9. n = 4 replicates per group per time-point, experiment repeated three times. e Cell cycle analysis in ATDC5 cells with or without Chmp5 deletion. n = 3 replicates per group, results repeated twice. f Alizarin red staining, von Kossa staining and alkaline phosphatase activity assay determining osteogenic activity in Chmp5 Ctsk compared to Chmp5 Ctsk/+ skeletal progenitors. n = 3 replicates each group, representative results of 3 mice per group. g Ingenuity pathway analysis of transcriptome data showing increased activity of osteoblast differentiation-related pathways in Chmp5 Ctsk compared to Chmp5 Ctsk/+ skeletal progenitors. Gene lists seeing Supplementary Table 2 . h Alizarin red staining and AlarmaBlue assay demonstrating osteogenesis and proliferation activity respectively in MC3T3-E1 cells with or without Chmp5 deletion by lentiviral CRISPR/CAS9. n = 4 replicates per group for alizarin red staining, n = 12 per group for AlarmaBlue assay; experiments repeated twice. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Cell number counting and AlamarBlue assay determining cell proliferation in Chmp5 Ctsk and Chmp5 Ctsk/+ skeletal progenitors at 2 weeks of age. n = 3 replicates per group per time-point; experiments repeated 3 times using cells from 3 mice. b Gene set enrichment analysis (GSEA) of transcriptome data showing positive enrichment of genes associated with cell senescence in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Enriched gene list referring to Supplementary Table 1 . n = 3 mice per group for RNA-sequencing analysis. c Gross image demonstrating phenotypes of accelerating senescence in Chmp5 Ctsk mice compared with Chmp5 Ctsk/+ mice at one year of age. Images are representative of 5 animals per group. d Cell number counting determining cell proliferation in ATDC5 cells with or without Chmp5 deletion by lentiviral CRISPR/CAS9. n = 4 replicates per group per time-point, experiment repeated three times. e Cell cycle analysis in ATDC5 cells with or without Chmp5 deletion. n = 3 replicates per group, results repeated twice. f Alizarin red staining, von Kossa staining and alkaline phosphatase activity assay determining osteogenic activity in Chmp5 Ctsk compared to Chmp5 Ctsk/+ skeletal progenitors. n = 3 replicates each group, representative results of 3 mice per group. g Ingenuity pathway analysis of transcriptome data showing increased activity of osteoblast differentiation-related pathways in Chmp5 Ctsk compared to Chmp5 Ctsk/+ skeletal progenitors. Gene lists seeing Supplementary Table 2 . h Alizarin red staining and AlarmaBlue assay demonstrating osteogenesis and proliferation activity respectively in MC3T3-E1 cells with or without Chmp5 deletion by lentiviral CRISPR/CAS9. n = 4 replicates per group for alizarin red staining, n = 12 per group for AlarmaBlue assay; experiments repeated twice. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Techniques: Alamar Blue Assay, RNA Sequencing Assay, CRISPR, Cell Cycle Assay, Staining, ALP Activity Assay, Activity Assay, Two Tailed Test

a Cell number counting and AlamarBlue assay determining cell proliferation in Ctsk + suture progenitors isolated from Chmp5 Ctsk ;Rosa26 mTmG/+ or Chmp5 Ctsk/+ ;Rosa26 mTmG/+ mice at 2 weeks of age. n = 3 replicates per group per time-point for cell number counting, n = 12 replicates per group for Alamarblue assay; representative results of 3 animals for each group. b Colony formation assay for Dmp1 + osteoprogenitors isolated from bones of Chmp5 Dmp1 ;Rosa26 mTmG/+ or Chmp5 Dmp1/+ ;Rosa26 mTmG/+ mice at 3 weeks of age. n = 5 animals per group. c TUNEL staining determining cell apoptosis in craniofacial sutures of Chmp5 Ctsk and Chmp5 Ctsk/+ mice. n = 3 animals per group; scale bars, 100 µm. d Annexin V stain examining cell apoptosis in Chmp5 Ctsk and Chmp5 Ctsk/+ skeletal progenitors. n = 3 replicates each group; repeated twice using cells from 2 mice per group. e Western blotting showing deletion of Chmp5 in ATDC5 cells by lentiviral CRISPR/CAS9. f AlamarBlue assay determining cell proliferation in ATDC5 cells with or without Chmp5 deletion. n = 12 replicates per group, repeated 3 times. g Annexin V-PI stain analyzing cell apoptosis in ATDC5 cells with or without Chmp5 deletion. n = 3-5 replicates each group, repeated 3 times. h Cell number counting examining cell proliferation in ATDC5 cells with or without Chmp5 deletion after treatment with the pan-caspase inhibitor Q-VD-OPh. n = 3 replicates each group each dose, repeated twice. i Quantitative PCR determining expression of Col2a1 and Sox9 in Chmp5 Ctsk and Chmp5 Ctsk/+ skeletal progenitors. n = 6 mice per group. J PANTHER GO-BP over-representation analysis of transcriptome data showing decreased activity of endochondral ossification-related pathways in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Minus (-), under-represented terms; n = 3 mice per group for RNA-sequencing analysis. k Western blotting and alizarin red staining respectively showing deletion of Chmp5 in mouse MC3T3-E1 cells by lentiviral CRISPR/CAS9 and increased activity of mineralization in Chmp5- deficient versus Chmp5 -sufficient MC3T3-E1 cells when induced toward osteogenesis. n = 4 replicates per group, repeated twice. All data are mean ± s.d.; 2way ANOVA followed by Sidak’s test for multiple comparisons or two-tailed Student’s t -test for 2-group comparison.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Cell number counting and AlamarBlue assay determining cell proliferation in Ctsk + suture progenitors isolated from Chmp5 Ctsk ;Rosa26 mTmG/+ or Chmp5 Ctsk/+ ;Rosa26 mTmG/+ mice at 2 weeks of age. n = 3 replicates per group per time-point for cell number counting, n = 12 replicates per group for Alamarblue assay; representative results of 3 animals for each group. b Colony formation assay for Dmp1 + osteoprogenitors isolated from bones of Chmp5 Dmp1 ;Rosa26 mTmG/+ or Chmp5 Dmp1/+ ;Rosa26 mTmG/+ mice at 3 weeks of age. n = 5 animals per group. c TUNEL staining determining cell apoptosis in craniofacial sutures of Chmp5 Ctsk and Chmp5 Ctsk/+ mice. n = 3 animals per group; scale bars, 100 µm. d Annexin V stain examining cell apoptosis in Chmp5 Ctsk and Chmp5 Ctsk/+ skeletal progenitors. n = 3 replicates each group; repeated twice using cells from 2 mice per group. e Western blotting showing deletion of Chmp5 in ATDC5 cells by lentiviral CRISPR/CAS9. f AlamarBlue assay determining cell proliferation in ATDC5 cells with or without Chmp5 deletion. n = 12 replicates per group, repeated 3 times. g Annexin V-PI stain analyzing cell apoptosis in ATDC5 cells with or without Chmp5 deletion. n = 3-5 replicates each group, repeated 3 times. h Cell number counting examining cell proliferation in ATDC5 cells with or without Chmp5 deletion after treatment with the pan-caspase inhibitor Q-VD-OPh. n = 3 replicates each group each dose, repeated twice. i Quantitative PCR determining expression of Col2a1 and Sox9 in Chmp5 Ctsk and Chmp5 Ctsk/+ skeletal progenitors. n = 6 mice per group. J PANTHER GO-BP over-representation analysis of transcriptome data showing decreased activity of endochondral ossification-related pathways in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Minus (-), under-represented terms; n = 3 mice per group for RNA-sequencing analysis. k Western blotting and alizarin red staining respectively showing deletion of Chmp5 in mouse MC3T3-E1 cells by lentiviral CRISPR/CAS9 and increased activity of mineralization in Chmp5- deficient versus Chmp5 -sufficient MC3T3-E1 cells when induced toward osteogenesis. n = 4 replicates per group, repeated twice. All data are mean ± s.d.; 2way ANOVA followed by Sidak’s test for multiple comparisons or two-tailed Student’s t -test for 2-group comparison.

Techniques: Alamar Blue Assay, Isolation, Colony Assay, TUNEL Assay, Staining, Western Blot, CRISPR, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, RNA Sequencing Assay, Two Tailed Test

a GSEA of transcriptome data showing positive enrichment of genes associated with the senescence-associated secretory phenotype in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Enriched gene list referring to Supplementary Table 1 . b Confocal images demonstrating extracellular vesicles (arrowheads) around Chmp5 Ctsk skeletal progenitors. Images are representative of 30 cells from 3 animals per group. Scale bars, 10 µm. c - e Nanoparticle tracking analysis of extracellular vesicles in culture media of Chmp5 Ctsk and wild-type skeletal progenitors. Data pooled from 3 replicates per group, 5 reads for each; repeated twice using cells from 2 mice per group. f Cell number counting determining cell proliferation in ATDC5 cells after treatment with culture supernatant from Chmp5 -deficient or Chmp5 -sufficient cells. n = 3 replicates per group per time-point, repeated twice. g , h Cell number counting, AlarmaBlue assay, and alizarin red staining analyzing cell proliferation ( g ) and osteogenesis ( h ) in neighboring CD45 - ;CD31 - ;GFP - progenitors sorted from Chmp5 Ctsk ;Rosa26 mTmG/+ or Ctsk- Cre;Rosa26 mTmG/+ mice. n = 3 replicates per group per time-point for cell number counting and alizarin red staining, n = 12 replicates per group for AlarmaBlue assay; repeated 3 times using cells from 3 animals. i Immunostaining of cell proliferation marker Ki-67 in periskeletal tissues around the knee of Chmp5 Ctsk and Chmp5 Ctsk/+ mice. n = 3 animals per group; dot-line representing the approximate border between the periskeletal overgrowth and femoral condyle; scale bars, 100 µm. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a GSEA of transcriptome data showing positive enrichment of genes associated with the senescence-associated secretory phenotype in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Enriched gene list referring to Supplementary Table 1 . b Confocal images demonstrating extracellular vesicles (arrowheads) around Chmp5 Ctsk skeletal progenitors. Images are representative of 30 cells from 3 animals per group. Scale bars, 10 µm. c - e Nanoparticle tracking analysis of extracellular vesicles in culture media of Chmp5 Ctsk and wild-type skeletal progenitors. Data pooled from 3 replicates per group, 5 reads for each; repeated twice using cells from 2 mice per group. f Cell number counting determining cell proliferation in ATDC5 cells after treatment with culture supernatant from Chmp5 -deficient or Chmp5 -sufficient cells. n = 3 replicates per group per time-point, repeated twice. g , h Cell number counting, AlarmaBlue assay, and alizarin red staining analyzing cell proliferation ( g ) and osteogenesis ( h ) in neighboring CD45 - ;CD31 - ;GFP - progenitors sorted from Chmp5 Ctsk ;Rosa26 mTmG/+ or Ctsk- Cre;Rosa26 mTmG/+ mice. n = 3 replicates per group per time-point for cell number counting and alizarin red staining, n = 12 replicates per group for AlarmaBlue assay; repeated 3 times using cells from 3 animals. i Immunostaining of cell proliferation marker Ki-67 in periskeletal tissues around the knee of Chmp5 Ctsk and Chmp5 Ctsk/+ mice. n = 3 animals per group; dot-line representing the approximate border between the periskeletal overgrowth and femoral condyle; scale bars, 100 µm. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Techniques: Staining, Immunostaining, Marker, Two Tailed Test

a Nanoparticle tracking analysis showing size distribution of extracellular vesicles in Chmp5 Ctsk and wild-type skeletal progenitors. Data pooled from 3 replicates, 5 reads for each; repeated twice using cells from 2 mice; D10, D50, and D90 indicating percent undersize, for example D50 = 229 nm representing that 50% vesicles are 229 nm or smaller. b, c Nanoparticle tracking analysis in ATDC5 cells with or without Chmp5 deletion. Data pooled from 2 replicates, 5 reads for each; repeated twice. d AlamarBlue assay determining activity of cell proliferation in ATDC5 cells after treatment with conditioned medium from Chmp5 -sufficient or Chmp5 -deficient cells. n = 12 replicates each group, repeated 3 times. e Immunostaining of Ki-67 in proximal tibia of the knee in Chmp5 Ctsk versus Chmp5 Ctsk/+ mice. Dot-lines representing the approximate border between perichondrial tissues and the tibial growth-plate. n = 3 animals per group; scale bars 100 µm. f Upregulated genes, coding secretory factors that potentially promote osteogenesis via a paracrine mechanism, in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 3 mice per group for RNA-sequencing analysis. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Nanoparticle tracking analysis showing size distribution of extracellular vesicles in Chmp5 Ctsk and wild-type skeletal progenitors. Data pooled from 3 replicates, 5 reads for each; repeated twice using cells from 2 mice; D10, D50, and D90 indicating percent undersize, for example D50 = 229 nm representing that 50% vesicles are 229 nm or smaller. b, c Nanoparticle tracking analysis in ATDC5 cells with or without Chmp5 deletion. Data pooled from 2 replicates, 5 reads for each; repeated twice. d AlamarBlue assay determining activity of cell proliferation in ATDC5 cells after treatment with conditioned medium from Chmp5 -sufficient or Chmp5 -deficient cells. n = 12 replicates each group, repeated 3 times. e Immunostaining of Ki-67 in proximal tibia of the knee in Chmp5 Ctsk versus Chmp5 Ctsk/+ mice. Dot-lines representing the approximate border between perichondrial tissues and the tibial growth-plate. n = 3 animals per group; scale bars 100 µm. f Upregulated genes, coding secretory factors that potentially promote osteogenesis via a paracrine mechanism, in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 3 mice per group for RNA-sequencing analysis. All data are mean ± s.d.; two-tailed Student’s t -test for 2-group comparison or 2way ANOVA followed by Sidak’s test for multiple comparisons.

Techniques: Alamar Blue Assay, Activity Assay, Immunostaining, RNA Sequencing Assay, Two Tailed Test

a Representative confocal images demonstrating abnormally enlarged vesicles in Chmp5 Ctsk relative to wild-type skeletal progenitors. Abnormal cells identified by containing enlarged GFP + vesicles. n = 200 cells from three mice per group. b Schematic showing molecular markers utilized for analyzing the endocytic pathway. c Representative confocal images showing LAMP1 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 20 cells each group. d Quantification of LAMP1 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 93, 66 cells respectively. e Representative confocal images showing RAB7 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 15 cells per genotype. f Quantification of RAB7 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 94, 92 cells respectively. g Co-localization of LAMP1 and RAB7 in ATDC5 cells with or without Chmp5 depletion. n = 20 cells per group. h Transmission electron microscopy (TEM) showing accumulation of multivesicular body (MVB)-like (arrows) and lysosome-like (arrowheads) structures in Chmp5 Ctsk relative to wild-type skeletal progenitors. n = 30 cells per group. i Confocal live-cell images demonstrating delayed degradation of EGF-conjugate in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 10 cells each group per time-point. Data shown as mean ± s.d.; two-tailed Student’s t -test. Scale bars, 10 µm except in ( h ) as indicated.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Representative confocal images demonstrating abnormally enlarged vesicles in Chmp5 Ctsk relative to wild-type skeletal progenitors. Abnormal cells identified by containing enlarged GFP + vesicles. n = 200 cells from three mice per group. b Schematic showing molecular markers utilized for analyzing the endocytic pathway. c Representative confocal images showing LAMP1 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 20 cells each group. d Quantification of LAMP1 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 93, 66 cells respectively. e Representative confocal images showing RAB7 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 15 cells per genotype. f Quantification of RAB7 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 94, 92 cells respectively. g Co-localization of LAMP1 and RAB7 in ATDC5 cells with or without Chmp5 depletion. n = 20 cells per group. h Transmission electron microscopy (TEM) showing accumulation of multivesicular body (MVB)-like (arrows) and lysosome-like (arrowheads) structures in Chmp5 Ctsk relative to wild-type skeletal progenitors. n = 30 cells per group. i Confocal live-cell images demonstrating delayed degradation of EGF-conjugate in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 10 cells each group per time-point. Data shown as mean ± s.d.; two-tailed Student’s t -test. Scale bars, 10 µm except in ( h ) as indicated.

Techniques: Immunostaining, Transmission Assay, Electron Microscopy, Two Tailed Test

a Representative confocal fluorescence images and quantification of fluorescence intensity of LysoTracker Red DND-99 in Chmp5 Ctsk compared to wild-type skeletal progenitors. n = 11 replicates per group for quantitative analysis; repeated 3 times using cells from 3 mice. Scale bars, 50 µm. b Isotype controls for immunofluorescence staining. Scale bars, 20 µm. c, d Confocal images of LAMP1( c ) and RAB7 ( d ) immunostaining in ATDC5 cells with or without Chmp5 deletion. Scale bars, 20 µm ( c ), 10 µm ( d ). e Additional TEM images showing accumulation of MVB-like (arrows) and lysosome-like (arrowheads) structures in Chmp5- deficient skeletal progenitors. f-h Representative confocal images of immunostaining for early endosome marker EEA1 ( f ), cycling endosome marker RAB11 ( g ), and trans-Golgi network marker TGN38 ( h ) in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 30 cells per group. Scale bars, 10 µm ( e ), 20 µm ( f ), 25 µm ( g ). Data shown as mean ± s.d.; two-tailed Student’s t -test.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Representative confocal fluorescence images and quantification of fluorescence intensity of LysoTracker Red DND-99 in Chmp5 Ctsk compared to wild-type skeletal progenitors. n = 11 replicates per group for quantitative analysis; repeated 3 times using cells from 3 mice. Scale bars, 50 µm. b Isotype controls for immunofluorescence staining. Scale bars, 20 µm. c, d Confocal images of LAMP1( c ) and RAB7 ( d ) immunostaining in ATDC5 cells with or without Chmp5 deletion. Scale bars, 20 µm ( c ), 10 µm ( d ). e Additional TEM images showing accumulation of MVB-like (arrows) and lysosome-like (arrowheads) structures in Chmp5- deficient skeletal progenitors. f-h Representative confocal images of immunostaining for early endosome marker EEA1 ( f ), cycling endosome marker RAB11 ( g ), and trans-Golgi network marker TGN38 ( h ) in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 30 cells per group. Scale bars, 10 µm ( e ), 20 µm ( f ), 25 µm ( g ). Data shown as mean ± s.d.; two-tailed Student’s t -test.

Techniques: Fluorescence, Immunofluorescence, Staining, Immunostaining, Marker, Two Tailed Test

a GSEA of transcriptome data reporting positive enrichment of genes associated with oxidative stress-induced senescence in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Enriched gene list referring to Supplementary Table 1 . b Confocal fluorescence imaging showing intracellular ROS (indicated by CellROX Deep Red) and mitochondria (indicated by TMRE) in Chmp5 Ctsk versus wild-type skeletal progenitors. Images are representative of 30 cells per group. Scale bars, 15 µm. c Quantification of cellular oxidative stress by examining fluorescence intensity of CellROX Deep Red. n = 10 each group, results repeated twice. d Confocal fluorescence imaging mapping intracellular ROS (indicated by CellROX Deep Red) and mitochondria (indicated by MitoTracker Green) in ATDC5 cells with or without Chmp5 depletion. n = 20 cells each group; scale bars, 10 µm. e Flow cytometry quantifying CellROX Deep Red stain in ATDC5 cells with or without Chmp5 depletion. n = 3 per group, experiment repeated twice. f Quantification of TMRE fluorescence intensity in Chmp5 -sufficient and Chmp5 -deficient ATDC5 cells. n = 12 each group, repeated 3 times. g Western blotting demonstrating changes of mitochondrial OXPHOS proteins NDUF88, SDHB, MT-CO1, UQCRC2, and ATF5A in Chmp5 -deficient relative to Chmp5 -sufficient ATDC5 cells. Experiment repeated twice. h Cell number counting determining cell proliferation in galactose medium. The same number of cells was plated at day 0 and cultured for 7 days. n = 6 per group, repeated three times. i Seahorse cell mitochondrial stress test showing mitochondrial respiratory capacity and extracellular acidification rate in Chmp5 Ctsk relative to wild-type skeletal progenitors. Data pooled from cells of 3 mice, 8 replicates for each animal. j TEM images showing mitochondrial morphology in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 50 cells each group; scale bars, 200 nm. k GSEA of transcriptome data and qPCR demonstrating positive enrichment of genes associated with DNA damage-induced senescence and upregulation of DNA damage responsive genes H2afx and Trp53 respectively in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 3 mice per group for RNA-sequencing analysis; n = 6 animals per group for qPCR. Enriched gene list for GSEA referring to Supplementary Table 1 . All data shown as mean ± s.d.; two-tailed Student’s t -test.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a GSEA of transcriptome data reporting positive enrichment of genes associated with oxidative stress-induced senescence in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. Enriched gene list referring to Supplementary Table 1 . b Confocal fluorescence imaging showing intracellular ROS (indicated by CellROX Deep Red) and mitochondria (indicated by TMRE) in Chmp5 Ctsk versus wild-type skeletal progenitors. Images are representative of 30 cells per group. Scale bars, 15 µm. c Quantification of cellular oxidative stress by examining fluorescence intensity of CellROX Deep Red. n = 10 each group, results repeated twice. d Confocal fluorescence imaging mapping intracellular ROS (indicated by CellROX Deep Red) and mitochondria (indicated by MitoTracker Green) in ATDC5 cells with or without Chmp5 depletion. n = 20 cells each group; scale bars, 10 µm. e Flow cytometry quantifying CellROX Deep Red stain in ATDC5 cells with or without Chmp5 depletion. n = 3 per group, experiment repeated twice. f Quantification of TMRE fluorescence intensity in Chmp5 -sufficient and Chmp5 -deficient ATDC5 cells. n = 12 each group, repeated 3 times. g Western blotting demonstrating changes of mitochondrial OXPHOS proteins NDUF88, SDHB, MT-CO1, UQCRC2, and ATF5A in Chmp5 -deficient relative to Chmp5 -sufficient ATDC5 cells. Experiment repeated twice. h Cell number counting determining cell proliferation in galactose medium. The same number of cells was plated at day 0 and cultured for 7 days. n = 6 per group, repeated three times. i Seahorse cell mitochondrial stress test showing mitochondrial respiratory capacity and extracellular acidification rate in Chmp5 Ctsk relative to wild-type skeletal progenitors. Data pooled from cells of 3 mice, 8 replicates for each animal. j TEM images showing mitochondrial morphology in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 50 cells each group; scale bars, 200 nm. k GSEA of transcriptome data and qPCR demonstrating positive enrichment of genes associated with DNA damage-induced senescence and upregulation of DNA damage responsive genes H2afx and Trp53 respectively in Chmp5 Ctsk relative to Chmp5 Ctsk/+ skeletal progenitors. n = 3 mice per group for RNA-sequencing analysis; n = 6 animals per group for qPCR. Enriched gene list for GSEA referring to Supplementary Table 1 . All data shown as mean ± s.d.; two-tailed Student’s t -test.

Techniques: Fluorescence, Imaging, Flow Cytometry, Staining, Western Blot, Cell Culture, RNA Sequencing Assay, Two Tailed Test

a Representative confocal images of TMRE showing accumulation of mitochondria in Chmp5 Ctsk skeletal progenitors. n = 30 cells per group, scale bars, 25 µm. b Additional TEM images showing the morphology of mitochondria in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 50 cells per group; scale bars, 200 nm.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Representative confocal images of TMRE showing accumulation of mitochondria in Chmp5 Ctsk skeletal progenitors. n = 30 cells per group, scale bars, 25 µm. b Additional TEM images showing the morphology of mitochondria in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 50 cells per group; scale bars, 200 nm.

Techniques:

a Cell number counting and AlamarBlue assay determining cell proliferation in ATDC5 cells with or without Chmp5 depletion after treatment with the antioxidant N -Acetylcysteine (NAC). n = 3 or 12 each group for cell number counting or AlamarBlue assay respectively, results repeated 3 times. b Alizarin red staining showing mineralization activity after treatment with NAC in Chmp5 Ctsk versus wild-type skeletal progenitors under osteogenic induction for 3 weeks. n = 3 or 4 per group, result repeated 5 times. c Gross images and radiography demonstrating hindlimb abduction and periskeletal bone overgrowth (arrows) respectively in Chmp5 Ctsk mice in comparison with Chmp5 fl/fl mice after treatment with quercetin and dasatinib (Q + D) or the vehicle PEG400 weekly for 7 weeks. n = 8-10 mice per group. d, e Measurement of mouth-opening and micro-CT images showing the improvement of the temporomandibular joint lesion in Chmp5 Ctsk mice after treatment with Q + D. Arrows indicating bone overgrowth around the mandibular condyle. Scale bars, 1 mm. f Animal body weight after treatment with Q + D or the vehicle PEG400. n = 5 mice per group; data of female mice shown; similar results found in both genders. g Four-limb hanging time and forelimb pull strength in Chmp5 Dmp1 and Chmp5 fl/fl mice after treatment with Q + D or the vehicle PEG400. n = 12 animals per group from both genders for hanging time test, 7 male mice per group for forelimb pull strength test; similar trend of changes found in both genders for the forelimb pull strength. All data are mean ± s.d.; 2way ANOVA followed by Sidak’s test for multiple comparisons.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a Cell number counting and AlamarBlue assay determining cell proliferation in ATDC5 cells with or without Chmp5 depletion after treatment with the antioxidant N -Acetylcysteine (NAC). n = 3 or 12 each group for cell number counting or AlamarBlue assay respectively, results repeated 3 times. b Alizarin red staining showing mineralization activity after treatment with NAC in Chmp5 Ctsk versus wild-type skeletal progenitors under osteogenic induction for 3 weeks. n = 3 or 4 per group, result repeated 5 times. c Gross images and radiography demonstrating hindlimb abduction and periskeletal bone overgrowth (arrows) respectively in Chmp5 Ctsk mice in comparison with Chmp5 fl/fl mice after treatment with quercetin and dasatinib (Q + D) or the vehicle PEG400 weekly for 7 weeks. n = 8-10 mice per group. d, e Measurement of mouth-opening and micro-CT images showing the improvement of the temporomandibular joint lesion in Chmp5 Ctsk mice after treatment with Q + D. Arrows indicating bone overgrowth around the mandibular condyle. Scale bars, 1 mm. f Animal body weight after treatment with Q + D or the vehicle PEG400. n = 5 mice per group; data of female mice shown; similar results found in both genders. g Four-limb hanging time and forelimb pull strength in Chmp5 Dmp1 and Chmp5 fl/fl mice after treatment with Q + D or the vehicle PEG400. n = 12 animals per group from both genders for hanging time test, 7 male mice per group for forelimb pull strength test; similar trend of changes found in both genders for the forelimb pull strength. All data are mean ± s.d.; 2way ANOVA followed by Sidak’s test for multiple comparisons.

Techniques: Alamar Blue Assay, Staining, Activity Assay, Micro-CT

X-ray images of Chmp5 Ctsk mice after treatment with Q + D or the vehicle PEG400 for 7 weeks. Arrows indicating bone overgrowths.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: X-ray images of Chmp5 Ctsk mice after treatment with Q + D or the vehicle PEG400 for 7 weeks. Arrows indicating bone overgrowths.

Techniques:

a X-ray images of Chmp5 fl/fl control mice after treatment with Q + D or the vehicle PEG400 for 7 weeks. b Gross images showing hindlimb abduction in Chmp5 Dmp1 and Chmp5 fl/fl mice after treatment with Q + D or the vehicle PEG400. n = 12 animals per group.

Journal: bioRxiv

Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

doi: 10.1101/2020.08.03.233874

Figure Lengend Snippet: a X-ray images of Chmp5 fl/fl control mice after treatment with Q + D or the vehicle PEG400 for 7 weeks. b Gross images showing hindlimb abduction in Chmp5 Dmp1 and Chmp5 fl/fl mice after treatment with Q + D or the vehicle PEG400. n = 12 animals per group.

Techniques:

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